Extended Data Fig. 3: mAID-based inducible KD system successfully depleted TgSNF2h, TgSNF2L and TgRFTS.
From: An ISWI-related chromatin remodeller regulates stage-specific gene expression in Toxoplasma gondii
Depletion of TgSNF2L-mAID-HA (a-c) and TgSNF2h-mAID-HA (d-f) upon addition of IAA for 24 hours was monitored by IFA. Fixed and permeabilized parasites were probed with HA (red) to detect TgSNF2h- or TgSNF2L-mAID-HA and co-stained with GAP45 (green). After 24 hours of protein depletion, parasites displayed aberrant morphology (a and d). Violin plots quantify nuclear HA signal in IFA after IAA addition (100 nuclei per condition); statistical significance was determined using one-way ANOVA and Tukey’s test (b and e). Complete protein loss after 5 hours of IAA treatment was confirmed via Western blot analysis of whole-cell extracts, using HA for detection and HDAC3 as a loading control (c and f). g. Depletion of TgRFTS protein using the mAID system after IAA addition was measured by IFA. Fixed and permeabilized parasites were probed with HA (red) to detect TgRFTS-mAID-HA. h. Violin plots quantify nuclear HA signal in IFA after IAA addition (100 nuclei per condition); statistical significance was determined using one-way ANOVA and Tukey’s test. i. The effects of conditional depletion of TgSNF2L, TgSNF2h, and TgRFTS on the lytic cycle were determined by plaque assay. 500 parasites were grown for 7 days in the presence or absence of IAA. No plaques were obtained after IAA addition, confirming that both remodelers and TgRFTS protein are essential for T. gondii's asexual in vitro growth. The area of all plaques was measured to create violin plots, and statistical significance was evaluated using P values from unpaired Student’s t-tests.
